The aim of this research is to identify the products of chemical reaction of carcinogenic alkylating agents with nucleic acids and correlate the extent and type of alkylation with the biological effects. (C14) Ethyl nitrosourea, which preferentially ethylates the oxygens of phosphodiesters forming phosphotriesters, will be reacted with infective TMV-RNA, in vitro, and answers to the following questions will be sought: 1) How many phosphate alkylations/TMV-RNA molecule are lethal, 2) Does lethality result from formation of phosphotriesters per se or from subsequent chain breakage, 3) How stable are ribophosphotriesters. Nucleotides will be reacted with a variety of alkylating agents in order to study the rate and extent of phosphate and base alkylation as a function of type of reagent, pH, and specificity for base vs. phosphate alkylation. The information gained from these studies should aid in preparing polynucleotides containing phosphotriesters. Such modified polynucleotides will be studied in terms of their interaction with complementary polynucleotides and their ability to act as templates.